Phospholipase C-γ2 Couples Bruton's Tyrosine Kinase to the NF-κB Signaling Pathway in B Lymphocytes

Mutations in the gene encoding Bruton's tyrosine kinase (BTK) interfere with B cell proliferation and lead to an X-linked immunodeficiency in mice characterized by reduced B cell numbers. Recent studies have established that BTK transmits signals from the B cell antigen receptor (BCR) to transcription factor NF-κB, which in turn reprograms a set of genes required for normal B cell growth. We now demonstrate that induction of NF-κB via this pathway requires the intermediate action of the -γ2 isoform of phospholipase C (PLC-γ2), a potential phosphorylation substrate of BTK. Specifically, pharmacologic agents that block the action of either PLC-γ2 or its second messengers prevent BCR-induced activation of IκB kinase. Moreover, activation of NF-κB in response to BCR signaling is completely abolished in B cells deficient for PLC-γ2. Taken together, these findings strongly suggest that PLC-γ2 functions as an integral component of the BTK/NF-κB axis following BCR ligation. Interference with this NF-κB cascade may account for some of the B cell defects reported forplc-γ2 −/− mice, which develop an X-linked immunodeficiency-like phenotype. Mutations in the gene encoding Bruton's tyrosine kinase (BTK) interfere with B cell proliferation and lead to an X-linked immunodeficiency in mice characterized by reduced B cell numbers. Recent studies have established that BTK transmits signals from the B cell antigen receptor (BCR) to transcription factor NF-κB, which in turn reprograms a set of genes required for normal B cell growth. We now demonstrate that induction of NF-κB via this pathway requires the intermediate action of the -γ2 isoform of phospholipase C (PLC-γ2), a potential phosphorylation substrate of BTK. Specifically, pharmacologic agents that block the action of either PLC-γ2 or its second messengers prevent BCR-induced activation of IκB kinase. Moreover, activation of NF-κB in response to BCR signaling is completely abolished in B cells deficient for PLC-γ2. Taken together, these findings strongly suggest that PLC-γ2 functions as an integral component of the BTK/NF-κB axis following BCR ligation. Interference with this NF-κB cascade may account for some of the B cell defects reported forplc-γ2 −/− mice, which develop an X-linked immunodeficiency-like phenotype. B cell antigen receptor X-linked immunodeficiency Bruton's tyrosine kinase phospholipase C-γ2 nuclear factor-κB IκB kinase protein kinase C phorbol 12-myristate 13-acetate bisindolylmaleimide I cyclosporin A 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester glutathione S-transferase electrophoretic mobility shift assays polyacrylamide gel electrophoresis mitogen-activated protein kinase The generation and survival of B lymphocyte subpopulations is contingent upon the expression of a functional B cell antigen receptor complex (BCR)1 (1Lam K.P. Kuhn R. Rajewsky K. Cell. 1997; 90: 1073-1083Abstract Full Text Full Text PDF PubMed Scopus (937) Google Scholar, 2Rajewsky K. Nature. 1996; 381: 751-758Crossref PubMed Scopus (1387) Google Scholar). BCR engagement directs B cell biological responses by initiating biochemical signaling cascades involving the cytoplasmic protein tyrosine kinases Lyn, Syk, and BTK (3Fruman D.A. Satterthwaite A.B. Witte O.N. Immunity. 2000; 13: 1-3Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 4Yang W.C. Collette Y. Nunes J.A. Olive D. Immunity. 2000; 12: 373-382Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 5Campbell K.S. Curr. Opin. Immunol. 1999; 11: 256-264Crossref PubMed Scopus (184) Google Scholar). BTK plays an integral role in transducing BCR-directed signals, because mutations in thebtk gene result in the B cell deficiencies X-linked agammaglobulinemia (XLA) in man and X-linked immunodeficiency (xid) in mice (6Vetrie D. Vorechovsky I. Sideras P. Holland J. Davies A. Flinter F. Hammarstrom L. Kinnon C. Levinsky R. Bobrow M.,. Smith C.I. Bently D.R. Nature. 1993; 361: 226-233Crossref PubMed Scopus (1253) Google Scholar, 7Tsukada S. Saffran D.C. Rawlings D.J. Parolini O. Allen R.C. Klisak I. Sparkes R.S. Kubagawa H. Mohandas T. Quan S. Belmont B.W. Cooper M.D. Conley M.E. Witte O.N. Cell. 1993; 72: 279-290Abstract Full Text PDF PubMed Scopus (1157) Google Scholar, 8Thomas J.D. Sideras P. Smith C.I. Vorechovsky I. Chapman V. Paul W.E. Science. 1993; 261: 355-358Crossref PubMed Scopus (573) Google Scholar, 9Rawlings D.J. Saffran D.C. Tsukada S. Largaespada D.A. Grimaldi J.C. Cohen L. Mohr R.N. Bazan J.F. Howard M. Copeland N.G. Jenkins N.A. Witte O.N. Science. 1993; 261: 358-361Crossref PubMed Scopus (777) Google Scholar, 10Khan W.N. Alt F.W. Gerstein R.M. Malynn B.A. Larsson I. Rathbun G. Davidson L. Muller S. Kantor A.B. Herzenberg L.A. Rosen F.S. Sideras P. Immunity. 1995; 3: 283-299Abstract Full Text PDF PubMed Scopus (640) Google Scholar). B cells from xid mice are defective in survival and proliferation, implicating BTK in these biological processes (10Khan W.N. Alt F.W. Gerstein R.M. Malynn B.A. Larsson I. Rathbun G. Davidson L. Muller S. Kantor A.B. Herzenberg L.A. Rosen F.S. Sideras P. Immunity. 1995; 3: 283-299Abstract Full Text PDF PubMed Scopus (640) Google Scholar, 11Anderson J.S. Teutch M. Dong Z. Wortis H.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10966-10971Crossref PubMed Scopus (116) Google Scholar, 12Solvason N. Wu W.W. Kabra N. Lund-Johansen F. Roncarolo M.G. Behrens T.W. Grillot D.A. Nunez G. Lees E. Howard M. J. Exp. Med. 1998; 187: 1081-1091Crossref PubMed Scopus (69) Google Scholar). However, the molecular mechanisms by which BTK effects B cell proliferation and survival are not well understood. Like BTK, transcription factor NF-κB has been implicated in the regulation of genes essential for B cell responses including proliferation and survival (13Bendall H.H. Sikes M.L. Ballard D.W. Oltz E.M. Mol. Immunol. 1999; 36: 187-195Crossref PubMed Scopus (57) Google Scholar, 14Grumont R.J. Rourke I.J. O'Reilly L.A. Strasser A. Miyake K. Sha W. Gerondakis S. J. Exp. Med. 1998; 187: 663-674Crossref PubMed Scopus (208) Google Scholar, 15Kontgen F. Grumont R.J. Strasser A. Metcalf D. Li R. Tarlinton D. Gerondakis S. Genes Dev. 1995; 9: 1965-1977Crossref PubMed Scopus (641) Google Scholar). In resting cells, NF-κB is sequestered in the cytoplasmic compartment via its association with a family of inhibitory proteins, termed IκBs (16Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5578) Google Scholar). Recent studies have identified a cytokine-inducible IκB kinase complex (IKK) consisting of two catalytic (IKKα and IKKβ) and one regulatory subunit (IKKγ) (17Zandi E. Karin M. Mol. Cell. Biol. 1999; 19: 4547-4551Crossref PubMed Scopus (307) Google Scholar). In response to NF-κB activating signals, IKK phosphorylates and targets IκB for degradation (17Zandi E. Karin M. Mol. Cell. Biol. 1999; 19: 4547-4551Crossref PubMed Scopus (307) Google Scholar). We and others (18Petro J.B. Rahman S.M. Ballard D.W. Khan W.N. J. Exp. Med. 2000; 191: 1745-1754Crossref PubMed Scopus (259) Google Scholar, 19Bajpai U.D. Zhang K. Teutsch M. Sen R. Wortis H.H. J. Exp. Med. 2000; 191: 1735-1744Crossref PubMed Scopus (190) Google Scholar) have recently shown that BTK couples the BCR to IKK and NF-κB. However, the biochemical mechanism by which BTK activates NF-κB remains largely undefined. BTK, in concert with the protein tyrosine kinase Syk and the adaptor protein BLNK, has recently been demonstrated to phosphorylate and activate PLC-γ2 (22Fluckiger A.C. Li Z. Kato R.M. Wahl M.I. Ochs H.D. Longnecker R. Kinet J.P. Witte O.N. Scharenberg A.M. Rawlings D.J. EMBO J. 1998; 17: 1973-1985Crossref PubMed Scopus (358) Google Scholar, 23Takata M. Homma Y. Kurosaki T. J. Exp. Med. 1995; 182: 907-914Crossref PubMed Scopus (183) Google Scholar, 24Takata M. Kurosaki T. J. Exp. Med. 1996; 184: 31-40Crossref PubMed Scopus (426) Google Scholar). In response to BCR signals, PLC-γ2 catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate, generating inositol 1,4,5-trisphosphate and diacylglycerol. Inositol 1,4,5-trisphosphate induces the release of Ca2+ from intracellular stores, and diacylglycerol facilitates the activation of PKC isoenzymes (20Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6175) Google Scholar, 21Imboden J.B. Stobo J.D. J. Exp. Med. 1985; 161: 446-456Crossref PubMed Scopus (574) Google Scholar). Thus, BTK-dependent activation of PLC-γ2 is essential for BCR-initiated calcium fluxes (22Fluckiger A.C. Li Z. Kato R.M. Wahl M.I. Ochs H.D. Longnecker R. Kinet J.P. Witte O.N. Scharenberg A.M. Rawlings D.J. EMBO J. 1998; 17: 1973-1985Crossref PubMed Scopus (358) Google Scholar). However, the functional consequences of PLC-γ2 signaling in the activation of nuclear factors that direct B cell responses are not known. In this report, we provide two lines of evidence indicating that BCR-initiated activation of NF-κB is mediated by PLC-γ2. First, DT40 chicken B cells deficient for PLC-γ2 fail to translocate NF-κB to the nucleus upon BCR activation. Second, pharmacologic inhibition of PLC-γ2 or its second messengers prevents BCR-responsive activation of IKK and phosphorylation of IκBα in primary B cells. These biochemical findings provide a potential molecular explanation for the B cell defects recently reported forplc-γ2 −/− mice, which display an xid-like phenotype reminiscent of animals lacking functional BTK (10Khan W.N. Alt F.W. Gerstein R.M. Malynn B.A. Larsson I. Rathbun G. Davidson L. Muller S. Kantor A.B. Herzenberg L.A. Rosen F.S. Sideras P. Immunity. 1995; 3: 283-299Abstract Full Text PDF PubMed Scopus (640) Google Scholar). The chicken B cell line DT40, DT40 cells deficient for either BTK or PLC-γ2 (DT40.BTK, DT40.PLC-γ2), or mutant DT40 cells reconstituted with either human BTK or PLC-γ2 (DT40.BTKR, DT40.PLC-γ2R) were a kind gift of Dr. Tomohiro Kurosaki, Riken Cell Bank, Japan (23Takata M. Homma Y. Kurosaki T. J. Exp. Med. 1995; 182: 907-914Crossref PubMed Scopus (183) Google Scholar, 24Takata M. Kurosaki T. J. Exp. Med. 1996; 184: 31-40Crossref PubMed Scopus (426) Google Scholar). DT40 cells were maintained as described previously and were cultured in low serum media (RPMI with 0.5% FCS, 0.05% chicken serum) for 8–12 h prior to stimulation (18Petro J.B. Rahman S.M. Ballard D.W. Khan W.N. J. Exp. Med. 2000; 191: 1745-1754Crossref PubMed Scopus (259) Google Scholar). Splenocytes and primary B lymphocytes were isolated from spleens of C57Bl6 mice. For phospho-IκBα Western analyses, RBC-depleted splenocytes were cultured and stimulated as indicated. For IKK in vitro kinase assays, B cells were purified by a process of negative selection on an affinity chromatography column (Cedarlane, Ontario, Canada). The purity of B cells isolated in this manner was ∼90–95% as verified by fluorescence-activated cell sorter analysis using anti-B220 and anti-IgM antibodies (PharMingen). All purifications were performed at 4 °C, and primary cells were used immediately upon purification. All pharmacological reagents were purchased from Calbiochem. For inhibition of BCR signaling, cells were incubated with EGTA (5 mm), BAPTA-AM (20 nm), cyclosporin A (20 μg/ml), bisindolylmaleimide I (20 μm), or U-73122 (5 μm) for 30 min prior to and during stimulation. Except where indicated in the figure legends, DT40 B cells were either left unstimulated or stimulated with a 1:2 dilution of hybridoma supernatants containing anti-chicken IgM monoclonal antibody (M4) or PMA and ionomycin, 1 μm each. Purified B cells (3–5 × 106 cells per sample) were incubated with 10 μg/ml polyclonal goat anti-mouse IgM F(ab′)2 fragments (Jackson ImmunoResearch), 10 μg/ml anti-mouse CD40 (PharMingen), or with PMA and ionomycin (1 μm each) at a cellular density of 2 × 106/ml in culture media (RPMI 1640 supplemented with 10% serum). To monitor any effects of serum on the activation of NF-κB, cells that were not stimulated were also incubated in medium containing 10% serum for the duration of stimulation. Nuclear extracts were prepared and used in DNA-binding reactions as described previously (18Petro J.B. Rahman S.M. Ballard D.W. Khan W.N. J. Exp. Med. 2000; 191: 1745-1754Crossref PubMed Scopus (259) Google Scholar). For EMSAs, an [α-32P]CTP- and [α-32P]ATP-labeled double-stranded oligonucleotide probe derived from the κB enhancer element of the IL-2Rα receptor promoter (5′-CAACGGCAGGGGAATTCCCCTCTCCTT-3′) was used. To verify equal amounts and integrity of proteins in the nuclear extracts, a control oligonucleotide for NF-Y was used. DNA-binding reactions were resolved by PAGE and visualized by autoradiography. For Western blot analysis of RelA and c-Rel, nuclear extracts equivalent to 2 × 107 cells were denatured in Laemmli reducing buffer by boiling at 95 °C for 3 min, and the proteins were resolved by SDS-PAGE. Proteins were electrotransferred onto nitrocellulose membranes and subjected to immunoblotting with rabbit polyclonal antibodies against RelA, c-Rel, or SP1 as described previously (18Petro J.B. Rahman S.M. Ballard D.W. Khan W.N. J. Exp. Med. 2000; 191: 1745-1754Crossref PubMed Scopus (259) Google Scholar). For IκBα degradation assays, 4 × 106cells/sample were preincubated for 30 min in medium containing 50 μm cycloheximide and then stimulated as indicated. Cell extracts were resolved by SDS-PAGE, transferred onto nitrocellulose membranes, probed with antibodies against chicken IκBα (pp40; gift of C. Chen) and p38 MAPK (Santa Cruz Biotechnology), and detected using the ECL system. Western blot analyses of IκBα phosphorylation were performed as above and probed with antibodies against mouse IκBα (Santa Cruz Biotechnology) or phosphorylated Ser-32/Ser-36 IκBα (Santa Cruz Biotechnology). The κB reporter plasmid encoding firefly luciferase under the control of a promoter containing six consensus NF-κB binding sites (6κB) and a control vector containing a Renilla luciferase gene fused to a thymidine kinase promoter have been described previously (25Chen C.L. Yull F.E. Kerr L.D. Biochem. Biophys. Res. Commun. 1999; 257: 798-806Crossref PubMed Scopus (8) Google Scholar). The indicated DT40 cell lines were each cotransfected by electroporation (250 V, 960 microfarads, Bio-Rad Gene Pulser) with 5 μg of the 6κB reporter construct and 1 μg of theRenilla construct. 18 h post-transfection, cells were stimulated for 6 h with anti-IgM. Cells were harvested, and levels of both firefly and Renilla luciferase were determined using a Dual Luciferase Reporter Assay System (Promega). Levels of firefly luciferase expression were normalized against Renilla as a control for transfection efficiency. In vitro kinase assays were performed on the cytosolic fraction of 5 × 106 B cells as described previously (18Petro J.B. Rahman S.M. Ballard D.W. Khan W.N. J. Exp. Med. 2000; 191: 1745-1754Crossref PubMed Scopus (259) Google Scholar). Briefly, cell extracts from 0.5 × 106 cell equivalents were removed for Western blot analysis, and the remaining cell extract was subjected to immunoprecipitation with anti-IKKα plus anti-IKKβ antibodies (Santa Cruz Biotechnology). The immunocomplexes were then resuspended in 20 μl of kinase buffer (20 mm HEPES, pH 7.2, MgCl2 (2 mm), MnCl2 (2 mm), dithiothreitol (1 mm), (20 containing of and 50 μg/ml The was to for 30 min at 30 °C under and then was by the of The were resolved by and with to the The were and to to In prior we established that BTK is required for nuclear of NF-κB in B cells (18Petro J.B. Rahman S.M. Ballard D.W. Khan W.N. J. Exp. Med. 2000; 191: 1745-1754Crossref PubMed Scopus (259) Google Scholar). However, the molecular mechanism by which BTK facilitates NF-κB activation is Recent findings suggest that BCR-directed nuclear of NF-κB requires the activation of the L. Sen R. J. Exp. Med. 1995; PubMed Scopus Google Scholar). To the mechanism by BTK to NF-κB we a role for calcium and in BCR-responsive nuclear of NF-κB in DT40 B cells. The DT40 B cell is to and has for biochemical analysis of T. Annu. Rev. Immunol. 1999; 17: PubMed Scopus Google Scholar). To calcium and a role in BCR-responsive activation of NF-κB in this cellular analyses were performed on nuclear extracts prepared from DT40 cells preincubated with pharmacological of and We used to and calcium and cyclosporin A to the or bisindolylmaleimide a of PKC isoenzymes We also DT40 cells with PMA and ionomycin, pharmacological agents to activate NF-κB via as a control A. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). BCR or in the nuclear of NF-κB 1 with 2 However, BCR-directed nuclear of NF-κB was by with or 3 and I not this response However, with in with either or in a block in NF-κB nuclear upon BCR activation 6 and result that inhibition of either calcium or and PKC completely BCR-directed activation of NF-κB in DT40 B cells. BCR BTK activates a set of to signaling (3Fruman D.A. Satterthwaite A.B. Witte O.N. Immunity. 2000; 13: 1-3Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, A.B. Li Z. Witte O.N. Immunol. 1998; PubMed Scopus Google Scholar). and PLC-γ2 have the to activate NF-κB via both and MAPK have been to IKK activation O.N. L.D. J.A. Nature. 1999; PubMed Scopus Google Scholar, J.A. Nature. 1999; PubMed Scopus Google a role has not been demonstrated for PLC-γ2. However, that calcium and PKC are essential for nuclear of NF-κB in DT40 cells PLC-γ2 in this response we an of PLC-γ2 in NF-κB nuclear in B cells stimulated via the To PLC-γ2 is for BCR-directed nuclear of NF-κB, we used mutant chicken DT40 B cells lacking PLC-γ2 with and DT40 B cells. Cells were via the and nuclear NF-κB was by BCR to a in nuclear NF-κB in DT40 cells 2 1 both and B cells to demonstrate this response 2 2 and 5 and 3 and However, PMA and ionomycin levels of nuclear NF-κB in cell 2 These strongly suggest that BTK, PLC-γ2 plays an essential role in the of BCR signals to activate NF-κB. To the was to of NF-κB we BCR-responsive nuclear of NF-κB in cells with that in DT40 B cells a of 4 h 2 BCR DT40 B cells NF-κB to the nucleus and maintained levels to 4 h activation. In nuclear levels of NF-κB not in B cells at any that 2 and with and To verify that the NF-κB activation in B cells was to PLC-γ2 were In response to BCR B cells human PLC-γ2 (23Takata M. Homma Y. Kurosaki T. J. Exp. Med. 1995; 182: 907-914Crossref PubMed Scopus (183) Google were of NF-κB nuclear as determined by and a NF-κB luciferase reporter C These strongly suggest that PLC-γ2 is for of signals that lead to the nuclear of NF-κB. of the family of proteins RelA, c-Rel, and which have the to either or (16Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5578) Google Scholar). NF-κB containing the family proteins RelA or have been demonstrated to the in response to BCR engagement in B cells We previously demonstrated that RelA and fail to nuclear upon BCR stimulation in B cells. To RelA and nuclear requires we the of and DT40 B cells to translocate these to the nucleus upon A and of nuclear extracts from anti-IgM stimulated and cells with antibodies that nuclear of both RelA and in DT40 B cells following BCR stimulation A and 1 and In BCR-responsive nuclear of RelA and is not in either or B cells. with nuclear of both A and in cell the in subunit are not to either in protein of the nuclear extracts or because amounts of the transcription factor SP1 are in A and Thus, BCR-directed nuclear of RelA and is NF-κB are in the of cells, to of a family of inhibitory termed BCR-induced nuclear of NF-κB is contingent upon the phosphorylation and degradation of a process that requires BTK. We the of B cells with and DT40 B cells to IκBα in response to BCR activation. Cells were incubated with anti-IgM antibodies or PMA and ionomycin for indicated and cytoplasmic extracts were for chicken IκBα DT40 B cells IκBα upon BCR activation. with the shown in B cells to IκBα in response to BCR stimulation with All cell lines IκBα in response to with PMA and of PLC-γ2 not the for IκBα These demonstrate that BCR-directed degradation of IκBα requires PLC-γ2. biochemical studies have identified NF-κB that on and including and (17Zandi E. Karin M. Mol. Cell. Biol. 1999; 19: 4547-4551Crossref PubMed Scopus (307) Google Scholar). we have recently established that BCR-initiated activation of NF-κB by BTK via IKK (18Petro J.B. Rahman S.M. Ballard D.W. Khan W.N. J. Exp. Med. 2000; 191: 1745-1754Crossref PubMed Scopus (259) Google Scholar). To that PLC-γ2 is required for BCR-directed nuclear of NF-κB, we a role for PLC-γ2 in IKK activation. We pharmacological agents that block PLC-γ2 and its second messengers prevent BCR-induced activation of IKK in primary B cells In response to activation signals via the BCR or or with IKK was as determined by in vitro kinase assays using as the substrate 5 1 with and In of B cells with either the or of its second messengers or prior to BCR stimulation abolished this 5 These and PKC in IKK activation upon BCR ligation. Moreover, verify the role of these signaling in BCR-responsive activation of IKK in a To this we performed Western blot analyses of cytosolic from or splenocytes using an antibody against IκBα 5 via either the BCR or CD40 phosphorylation of IκBα 5 and BCR-responsive IκBα phosphorylation was by with either or I 5 PMA stimulation in IκBα phosphorylation that was by with the PKC I 5 and These and PKC in BCR-responsive activation of IKK and phosphorylation of Moreover, the that cells with fail to activate IKK upon BCR as a of this is with the that and to IKK activation in cells A. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). these suggest that PLC-γ2 is to BCR-responsive activation of phosphorylation of and nuclear of NF-κB. We have that PLC-γ2 and its signals are essential for BCR-directed activation of IKK and NF-κB. studies in B cells have that PLC-γ2 is via the of BTK, Syk, and (3Fruman D.A. Satterthwaite A.B. Witte O.N. Immunity. 2000; 13: 1-3Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, D.J. Immunol. 1999; PubMed Scopus Google Scholar). is that PLC-γ2 is the BTK for BCR-directed activation of IKK and NF-κB. is required to BTK including and with PLC-γ2 to nuclear of NF-κB in B cells. However, the of PLC-γ2 in the signaling pathway the potential molecular explanation for the xid-like B cell deficiencies −/− −/− mice (10Khan W.N. Alt F.W. Gerstein R.M. Malynn B.A. Larsson I. Rathbun G. Davidson L. Muller S. Kantor A.B. Herzenberg L.A. Rosen F.S. Sideras P. Immunity. 1995; 3: 283-299Abstract Full Text PDF PubMed Scopus (640) Google Scholar, D. J. R. J.C. E. A. Immunity. 2000; 13: Full Text Full Text PDF PubMed Scopus Google Scholar). We for and and for in the of this

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